RESUMEN
Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat-shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii, and behave as psychrophile and psychrotroph micro-organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell's somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 degrees C, a much smaller increase was revealed in heat-shocked cells of E. focardii. These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.
Asunto(s)
Euplotes/genética , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Animales , Regiones Antárticas , Fraccionamiento Celular , ADN Protozoario/genética , Euplotes/fisiología , Genes Protozoarios , Proteínas HSP70 de Choque Térmico/metabolismo , Immunoblotting , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Especificidad de la EspecieRESUMEN
In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement.
Asunto(s)
ADN Protozoario/genética , Secuencias Reguladoras de Ácidos Nucleicos , Tetrahymena thermophila/genética , Animales , Núcleo Celular/metabolismo , ADN Protozoario/metabolismo , ADN Recombinante/genética , ADN Recombinante/metabolismo , Eliminación de Secuencia , Transformación GenéticaRESUMEN
In hypotrich ciliates, the entire silent chromosomal genome of the germinal nucleus (micronucleus) undergoes extensive DNA rearrangements that, during the development of the somatic nucleus (macronucleus) at the beginning a new cell life cycle, eventually result in the production of linear DNA molecules. These molecules represent functional genes, each one consisting of a central coding region flanked by two shorter regions, which apparently lack canonical elements for regulation of replication and transcription. These are amplified to thousands of copies in the "adult" macronucleus of the vegetative cell. We defined the extent of this amplification for allelic codominant genes which, in the macronucleus of Euplotes raikovi, encode polypeptide cell recognition factors (pheromones). This amplification was shown to be allele-specific. The copy numbers of genes coding for pheromones Er-1, Er-2, and Er-10 were determined to be 2.5-2.9 x 10(4), 0.9-1.2 x 10(4), 1.6-1.85 x 10(4) respectively, and these numbers did not appreciably vary during the vegetative cell proliferation. This differential amplification of pheromone genes was (i) independent of whether two genes coexisted in the same heterozygous cell or were separated in the corresponding homozygotes, and (ii) directly correlated with quantitative variations in mRNA synthesis and pheromone secretion. On the basis of these results, it is suggested that a mechanism of gene-specific amplification may be used by hypotrich ciliates to modulate gene expression.
Asunto(s)
Euplotes/genética , Amplificación de Genes , Proteínas de la Membrana/genética , Feromonas/genética , Proteínas Protozoarias/genética , Animales , Secuencia de Bases , Cartilla de ADN , Euplotes/metabolismo , Genes Protozoarios , Datos de Secuencia MolecularRESUMEN
In the ciliate Euplotes raikovi, the same cell that secretes the pheromone Er-1, a polypeptide of 40 amino acids derived from a precursor (prepro-Er-1) of 75 amino acids, also produces a polypeptide of 130 amino acids, of which the 75 residues at the carboxyl terminus are identical to those of prepro-Er-1 and the 55 residues at the amino terminus form a new sequence. This larger Er-1 isoform is retained in membranes, where it may function as a binding site for soluble Er-1 in a mechanism of autocrine secretion. Membrane-bound and soluble Er-1 are translated from two mRNAs that apparently originate from a common micronuclear and/or macronuclear gene through alternative elimination of intervening sequences. This finding suggests that single genes responsible for the generation of isoform diversity in polypeptide hormones are present even in single-celled eukaryotes.
Asunto(s)
Euplotes/genética , Proteínas de la Membrana/genética , Feromonas/genética , Proteínas Protozoarias/genética , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN/genética , Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química , Empalme del ARN , ARN Mensajero/genética , SolubilidadRESUMEN
The precursors of Euplotes raikovi pheromones Er-2 and Er-10 have been structurally characterized from the sequences of their coding regions that were amplified and cloned using the polymerase chain reaction and oligonucleotide primers corresponding to conserved sequences of the gene for pheromone Er-1. The predicted amino acid sequences contain 75 residues distributed through three domains: signal peptide, pro segment and mature pheromone. Despite the conservation of the overall length, there is variation in the size of the pro segments and of the mature pheromones. The comparison of the sequences shows a gradient of identity from the amino to the carboxyl terminus; the signal sequences are identical (with greater than or equal to 95% identity in the nucleotide sequences), the pro segments more variable and the mature pheromones quite diverse. The processing site of the pro pheromones, to produce the mature forms, is apparently characterized by the unusual Xaa-Asp sequence.
Asunto(s)
Euplotes/genética , Feromonas/genética , Precursores de Proteínas/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Euplotes/fisiología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN/genética , ARN/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Moldes GenéticosRESUMEN
cDNA clones comprising the entire coding region for the mating pheromone Er-1 of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors lambda gt10 and pUC12. The cDNA sequence contains an open reading frame of 75 amino acids that constitute pre-pro-Er-1. The amino acid sequence of secreted Er-1 starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-1 protein sequencing. The coding region of Er-1 cDNA ends with codon TAA, which specifies glutamine in other ciliates. The 5'- and 3'-noncoding regions contain, respectively, two and one inverted repeats. The 3'-noncoding-region inverted repeat, which includes the unusual polyadenylylation signal AACAAA, has been related to RNA 3'-terminus formation.